Nanosight Analysis

Overall Objective

Determine the concentration of total exosomes throughout pregnancy in WT mated C57/B6 mice. Time points for the study included Virgin (day 1), gestational day (G.D) 5.5, 10.5, 14.5, 17.5 and 1 day post partum (day 20). six animals were used for each of the six time points in the study. A volume of 100ul of plasma was obtained by means of cardiac puncture or deeply anesthetized animals. Plasma exosomes were isolated using the total exosome isolation reagent (ThermoFisher) and resuspended in 30ul of PBS. Samples were then blinded (randomly assigned numbers) and analzed by Nanosight (NS300 Malvern) nanoparticle tracking analysis. For each day of nanosight analysis, one sample from each of the time points was chosen and analyzed by Nanosight. Before and after samples were analyzed, 100nm polystyrene bead standards were analyzed (1:125 in PBS) to determine intra and inter assay variability.

Nanosight measurement/ experimental setup

Samples and standards were measured twice by two separate injections (from separate tubes) into the machine. Three, thirty second 30 second videos were recorded and analyzed by Nanosight NTA 3.2 and exported raw data was exported as a .csv file which was minimally processed for easy import into R.

Nanosight acquisition settings

	Gain	Level	Threshold
Standards	1	11	4
Samples	1	12	4

Load Libraries

library(tidyverse)
library(cowplot)
library(broom)
library(pwr)
library(plotly)

Import data

setwd("~/GitHub/time-course/data")
#setwd("~/Library/Mobile\ Documents/com~apple~CloudDocs/time-course/data")

rawdata <- "revised_MASTER-ExperimentSummary.csv"
timecourse <- "timecourse2017.csv"
standards <- "standards_MASTER-ExperimentSummary.csv"


data <- read_csv(rawdata)
tc <- read_csv(timecourse, na = c("","NA"))
std <- read_csv(standards)

Convert data from ‘wide’ to ‘long’ format

data1 <- data %>%
  gather(Sample,Count,2:250)

# Separate samples by identifiers 
data2 <- data1 %>% 
  separate(Sample, into=c("Sample_ID","Dilution_factor","Injection","Tech_rep", sep = "_")) %>% 
  select(-`_`)


std1 <- std %>% 
  gather(Sample,Count,2:82)

std2 <- std1 %>% 
  separate(Sample, into=c("Sample_ID","When","Dilution_factor","Nano_day","Injection","Tech_Rep", sep = "_")) %>% 
  select(-`_`)


std2$Sample_ID <- as.factor(std2$Sample_ID)
std2$When <- as.factor(std2$When)
std2$Dilution_factor <- as.numeric(std2$Dilution_factor)
std2$Injection<- as.factor(std2$Injection)
std2$Nano_day <- as.numeric(std2$Nano_day)

Backcalculate standards

std2 <- std2 %>% 
  mutate(True_Count=Dilution_factor*Count)

std2$Nano_day <-  factor(std2$Nano_day, levels=c('1','2','3','4','5','6','7'))
std2$When <- factor(std2$When, levels=c('before','after'))

Summarize three technical replicates

std3 <- std2 %>% 
  group_by(particle_size,Sample_ID,When,Dilution_factor,Nano_day,Injection) %>% 
  summarise( tech_N = length(True_Count),
             tech_mean = mean(True_Count),
             tech_sd = sd(True_Count),
             tech_se = tech_sd/sqrt(tech_N))
std3

Summarize by injection

std4 <- std3 %>% 
  group_by(Nano_day,When,particle_size) %>% 
  summarise( inj_N = length(tech_mean),
             inj_mean = mean(tech_mean),
             inj_sd = sd(tech_mean),
             inj_se = inj_sd/sqrt(inj_N))
std4

Plot before and after plots, facet by experimental day

std_day <- std4 %>% 
  ggplot(aes(x=particle_size,y=inj_mean,color=When))+
  geom_ribbon(aes(ymin=inj_mean-inj_se, ymax=inj_mean+inj_se),alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=2) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\n100nm standards")+ #title
  labs(color="Condition")+ #Label table title
  facet_grid(. ~ Nano_day)
std_day

###Plot facet by when and experimental day

std_day_facet <- std4 %>% 
  ggplot(aes(x=particle_size,y=inj_mean,color=When))+
  geom_ribbon(aes(ymin=inj_mean-inj_se, ymax=inj_mean+inj_se),alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=2) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\n100nm standards")+ #title
  labs(color="Condition")+ #Label table title
  facet_grid(When ~ Nano_day)
std_day_facet

## Warning: Removed 1000 rows containing missing values (geom_path).

Particle concentrations from each experimental day

std_df <- std4 %>% 
  group_by(Nano_day,When) %>% 
  summarise(total=sum(inj_mean))
std_df

Bar graph of particle concentrations

std_df %>% 
  ggplot(aes(x=Nano_day,y=total,fill=When))+
  geom_col(position="dodge")+
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Experimental Day") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\n100nm standards")+ #title
  labs(color="When") #Label table title

###Intraassay variability

Intra.assay_cv <- std_df %>% 
  group_by(Nano_day) %>% 
  summarise(Day_N = length(total),
             Day_mean = mean(total),
             Day_sd = sd(total),
             Day_se = Day_sd/sqrt(Day_N),
            Day_cv = Day_sd/Day_mean )
Intra.assay_cv

Inter assay variability

Inter.assay <- Intra.assay_cv %>% 
 summarise(Exp_N = length(Day_mean),
             Exp_mean = mean(Day_mean),
             Exp_sd = sd(Day_mean),
             Exp_se = Exp_sd/sqrt(Exp_N),
            Exp_cv = Exp_sd/Exp_mean )
Inter.assay

Factor the data into categorical variables

# Refactoring Columns for samples
data2$Sample_ID <- as.factor(data2$Sample_ID)
data2$Dilution_factor <- as.numeric(data2$Dilution_factor)
data2$Injection<- as.factor(data2$Injection)
data2$Tech_rep <- as.numeric(data2$Tech_rep)


# Refactoring COlumns for timecourse
tc$Sample_ID <- as.factor(tc$Sample_ID)
tc$Day <- as.factor(tc$Day)
tc$Weight <- as.numeric(tc$Weight)
tc$TEI_Day <- as.factor(tc$TEI_Day)

tc1 <- tc %>% 
  select(Day:Pups)
tc1

Back calculate the original concentration of the sample

data2 <- data2 %>% 
  mutate(True_Count=Dilution_factor*Count)
data2

Average the three technical replicate readings

data3 <- data2 %>% 
  group_by(particle_size,Sample_ID,Dilution_factor,Injection) %>% 
  summarise( tech_N = length(True_Count),
             tech_mean = mean(True_Count),
             tech_sd = sd(True_Count),
             tech_se = tech_sd/sqrt(tech_N))
data3

test1 <- left_join(tc1,data3, by= "Sample_ID")

Summarize samples by injection (average both injections)

data4 <- data3 %>% 
  group_by(particle_size,Sample_ID,Dilution_factor) %>% 
  summarise( inj_N = length(tech_mean),
             inj_mean = mean(tech_mean),
             inj_sd = sd(tech_mean),
             inj_se = inj_sd/sqrt(inj_N))
data4

test2 <- left_join(tc1,data4, by= "Sample_ID")

test2

Quick visualizations

Graphing all samples

test1$Sample_ID_correct = factor(test1$Sample_ID, levels=c('1','2','3','4','5','6','7','8','9','10','11','12','13','14','15','16','17','18','19','20','21','22','23','24','25','26','27','28','29','30','31','32','33','34','35','36','70','73','74','75'))

graph1 <- test1 %>%
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=2.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nMouse Plasma Throughout Pregnancy")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct, nrow=7)

graph1

Looking at individual gestational day nanosight line plots

Virgin Mice

test1 %>%
  filter(Day == '1') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nVirgin Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

GD 5.5

test1 %>%
  filter(Day == '5') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nGD 5.5 Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

GD 10.5

test1 %>%
  filter(Day == '10') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nGD 10.5 Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

GD 14.5

test1 %>%
  filter(Day == '14') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nGD 14.5 Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

GD 17.5

test1 %>%
  filter(Day == '17') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nGD 17.5 Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

1 Day Post

test1 %>%
  filter(Day == '20') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se,
                  ymax=tech_mean+tech_se),
                  alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\n1 Day Post Partum Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~ Sample_ID_correct)+
  geom_vline(xintercept = 140)+
  annotate("text", x= 235, y = 4E9, label= "140nm")

Graphing averaged samples by experimental day

graph2 <- test2 %>%
  group_by(TEI_Day) %>% 
  ggplot(aes(x=particle_size, y=inj_mean,color=Day ))+ #plot
  #geom_ribbon(aes(ymin=inj_mean-inj_se, ymax=inj_mean+inj_se),alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=2) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nMouse Plasma Throughout Pregnancy")+ #title
  labs(color="Condition")+ #Label table title
  facet_wrap(~ TEI_Day, ncol=7)

graph2

### Particle concentration values for each of the 36 samples

test3 <- test2 %>% 
  group_by(Day,Sample_ID) %>% 
  summarise(particle_conc=sum(inj_mean))
test3

Summary statistics of particle concentration (averaging n=6 for each time point)

test4 <- test3 %>% 
  filter(!Sample_ID %in% c('6','26','28','32')) %>% #Removing outliers
  group_by(Day) %>% 
  summarise(Day_N=length(particle_conc),
            Day_mean = mean(particle_conc),
            Day_sd = sd(particle_conc),
            Day_se = Day_sd/sqrt(Day_N))
test4

Boxplot

plot1 <- test3 %>% 
  filter(!Sample_ID %in% c('6','26','28','32')) %>% 
  group_by(Day) %>% 
  ggplot(aes(x= Day, y = particle_conc/1E9, color=Day)) +
  geom_boxplot(colour="black",fill=NA) + 
  geom_point(aes(text = paste("Sample ID:", Sample_ID)),
             position='jitter',size=3)+
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="Condition")+ # Label table title
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_color_discrete(labels=c("Virgin","5","10","14","17","1 Day Post")) # Change Legend
  
plot1

#ggsave("Exosome_plot.png", height = 5, width = 7, units = "in", dpi = 600)

Plotly

  ggplotly(plot1)

Bar plot

plot <- test4 %>% 
  ggplot(aes(x=Day, y=Day_mean, fill=Day))+ #plot
  geom_col()+
  geom_errorbar(aes(ymin=Day_mean-Day_se, ymax=Day_mean+Day_se), width=.5, 
                size=0.8, colour="black", position=position_dodge(.9)) + #error bars
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nMean Vesicles /ml\n") + # Y axis label
  ggtitle("\nPlasma Vesicle Concentration\nAcross Pregnancy\n")+ #title
  guides(fill=FALSE) + # Remove legend
  scale_y_continuous(breaks = seq(1E11,5E11,1E11),
                     limits = c(0,5E11),
                     expand=c(0,0))+ #set bottom of graph
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_fill_discrete(labels=c("Virgin","5","10","14","17","1 Day Post")) # Rename bottom


plot

ggsave("Vesicle_barplot.png", height = 5, width = 7, units = "in", dpi = 600)

Looking at Variation between the days the samples were run

test7 <- test3 %>% 
  left_join(tc1)

## Joining, by = c("Day", "Sample_ID")

plot2 <- test7 %>%
  ggplot(aes(x = Day, y = particle_conc, color = Day, shape=TEI_Day))+
  geom_point(position= 'dodge',size=4)+
  scale_shape_manual(values=c(15,16,17,18,22,23,24))+
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="Condition") + # Label table title
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_color_discrete(labels=c("Virgin","5","10","14","17","1 Day Post"))

plot2

ggplotly(plot2)

## Warning: Width not defined. Set with `position_dodge(width = ?)`

Statistics on ALL sizes

Shapiro test

tidy(shapiro.test(test3$particle_conc))

p value >0.05 therefore conclude data is normally distributed

ANOVA

jacob <-   test3 %>%  # Datset without outliers
  filter(!Sample_ID %in% c('6','26','32'))
         
fit <- aov(particle_conc ~ Day, data=jacob)
stats <- tidy(fit)
stats

Statistically significant, thus Tukey’s HSD post hoc analysis can determine significant differences.

Tukey Post Hoc Test

HSD <- TukeyHSD(fit)
tukey <- tidy(HSD)
tukey

Significant Tukey Post Hoc Test Values

tukey %>%
  filter(adj.p.value<0.05) %>% 
  arrange(adj.p.value)

Next we can filter on vesicles that are 140nm and smaller

nano_140 <- data4 %>% 
  filter(particle_size<140.5) %>% 
  left_join(tc1, by= "Sample_ID") %>% 
  group_by(Day,Sample_ID) %>% 
  summarise(particle_conc=sum(inj_mean)) %>% 
  filter(!Sample_ID %in% c('6','26','28','32'))

nano_140_plot <- nano_140 %>% 
  ggplot(aes(factor(Day),particle_conc, color=Day)) +
  geom_boxplot(colour="black",fill=NA) + 
  geom_point(aes(text = paste("Sample ID:", Sample_ID)),
             position='jitter',size=3)+
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nMean Exosomes /ml\n") + # Y axis label
  ggtitle("\nExosome Concentration\nAcross Pregnancy\n")+ #title
  guides(fill=FALSE) + # Remove legend
  scale_y_continuous(breaks = seq(1E11,5E11,1E11),
                     limits = c(0,5E11),
                     expand=c(0,0))+ #set bottom of graph
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_fill_discrete(labels=c("Virgin","5","10","14","17","1 Day Post")) # Rename bottom

nano_140_plot 

ggplotly(nano_140_plot)

nano_140_bar <- nano_140 %>% 
  group_by(Day) %>% 
  summarise(N    = length(particle_conc),
            mean = mean(particle_conc),
            sd   = sd(particle_conc),
            se   = sd/sqrt(N)) %>% 
  ggplot(aes(x= factor(Day),y = mean, fill=Day)) +
  geom_col()+
  geom_errorbar(aes(ymin=mean-se, ymax=mean+se), width=.5, 
                size=0.8, colour="black", position=position_dodge(.9)) + #error bars
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nMean Exosomes /ml\n") + # Y axis label
  ggtitle("\nPlasma Exosome Concentration\nAcross Pregnancy\n")+ #title
  guides(fill=FALSE) + # Remove legend
  scale_y_continuous(breaks = seq(1E11,5E11,1E11),
                     limits = c(0,4E11),
                     expand=c(0,0))+ #set bottom of graph
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_fill_discrete(labels=c("Virgin","5","10","14","17","1 Day Post")) # Rename bottom

nano_140_bar 

ggsave("Exosome_barplot.png", height = 5, width = 7, units = "in", dpi = 600)

Statistics on Exosomes only (140nm)

Shapiro test

tidy(shapiro.test(nano_140$particle_conc))

p value >0.05 therefore conclude data is normally distributed

ANOVA

exo_fit <- aov(particle_conc ~ Day, data=nano_140)
exo_stats <- tidy(exo_fit)
exo_stats

Statistically significant, thus Tukey’s HSD post hoc analysis can determine significant differences.

Tukey Post Hoc Test

exo_HSD <- TukeyHSD(exo_fit)
exo_tukey <- tidy(exo_HSD)
exo_tukey

Significant Tukey Post Hoc Test Values

exo_tukey %>%
  filter(adj.p.value<0.05) %>% 
  arrange(adj.p.value)

test8 <- test7 %>% 
  filter(!Day == "20")

fit <- lm(particle_conc ~ Weight ,data = test8)

summary(fit)

## 
## Call:
## lm(formula = particle_conc ~ Weight, data = test8)
## 
## Residuals:
##        Min         1Q     Median         3Q        Max 
## -2.600e+11 -7.122e+10 -2.456e+10  8.690e+10  2.239e+11 
## 
## Coefficients:
##              Estimate Std. Error t value Pr(>|t|)   
## (Intercept) 5.768e+10  7.666e+10   0.752  0.45727   
## Weight      8.548e+09  2.849e+09   3.000  0.00519 **
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 1.127e+11 on 32 degrees of freedom
## Multiple R-squared:  0.2196, Adjusted R-squared:  0.1952 
## F-statistic: 9.002 on 1 and 32 DF,  p-value: 0.00519

tidy(summary(fit))

test7 %>% 
  ggplot(aes(x= Weight, y = particle_conc))+
  geom_point()+
  geom_smooth()+
  xlab("\nWeight (g)\n") + # X axis label
  ylab("Exosomes/ml\n") + # Y axis label
  ggtitle("Linear Regression of Exosome \nConcentration vs. Weight")+ #title
  labs(color="Day")#Label table title

## `geom_smooth()` using method = 'loess'

test7 %>% 
  filter(!Sample_ID == '70') %>% 
  ggplot(aes(x= Weight, y = particle_conc))+
  geom_point(size = 3,aes(color=factor(Day)))+
  geom_smooth(method = "lm", level = 0.95)+
  xlab("\nWeight (g)\n") + # X axis label
  ylab("Exosomes/ml\n") + # Y axis label
  ggtitle("Linear Regression of Exosome \nConcentration vs. Weight")+ #title
  labs(color="Day")#Label table title

test7 %>% 
  ggplot(aes(x= Pups, y = particle_conc))+
  geom_point(size = 4, aes(color=factor(Day)))+
  #geom_smooth(method = "lm", level = 0.95)+
  scale_x_continuous(breaks=seq(0,12,2))+
  xlab("\nNumbwe of Pups\n") + # X axis label
  ylab("Exosomes/ml\n") + # Y axis label
  ggtitle("Linear Regression of Exosome \nConcentration vs. Number of Pups")+ #title
  labs(color="Day")#Label table title

mean_placenta <- tc %>% 
  filter(Day %in% c('10','14','17') & !Sample_ID %in% c('70','73','74','75')) %>%
  select(-(TEI_Day:Pup_right),-Resorp) %>% 
  gather("Placenta_avg","Plac_weight", 3:5) %>%
  group_by(Day,Sample_ID) %>% 
  summarise(N = length(Plac_weight),
            mean_plac = mean(Plac_weight*1000, na.rm = TRUE), #convert g to mg
            sd = sd(Plac_weight)*1000,  #convert g to mg
            se = sd/sqrt(N))

mean_placenta %>% 
  inner_join(test7) %>% 
  ggplot(aes(x= mean_plac, y = particle_conc))+
  geom_point(size= 3,aes(color=factor(Day)))+
  geom_smooth(method = "lm", level = 0.95)+
  xlab("\nPlacental Weight (mg)\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="G.D.")#Label table title

## Joining, by = c("Day", "Sample_ID")

## Warning in inner_join_impl(x, y, by$x, by$y, suffix$x, suffix$y): joining
## factor and character vector, coercing into character vector

mean_placenta %>% 
  inner_join(test7) %>% 
  ggplot(aes(x = Pups, y = particle_conc))+
  geom_point(size= 3,aes(color=factor(Day)))+
  geom_smooth(method = "lm", se= FALSE)+
  facet_wrap(~Day)+
  scale_x_continuous(breaks=seq(1,12,2))+
  xlab("\nNumbr of Pups\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="G.D.")#Label table title

## Joining, by = c("Day", "Sample_ID")

## Warning in inner_join_impl(x, y, by$x, by$y, suffix$x, suffix$y): joining
## factor and character vector, coercing into character vector

test3 %>% 
  filter(!Day %in% c('1','20')) %>% 
  group_by(Day) %>% 
  ggplot(aes(factor(Day),particle_conc, color=Day)) +
  geom_point(size=3)+
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="Condition")#Label table title

nanosight_plot <- test1 %>%
  filter(Sample_ID == '75') %>% 
  ggplot(aes(x=particle_size, y=tech_mean,color=Injection ))+ #plot
  geom_ribbon(aes(ymin=tech_mean-tech_se, ymax=tech_mean+tech_se),alpha=0.2,fill = alpha('grey12', 0.2)) + #error bars
  geom_line(size=1.0) + xlim(0,500)+ #line size, x-axis scale
  scale_y_continuous(expand=c(0,0))+ #set bottom of graph
  xlab("Particle Size (nm)") + # X axis label
  ylab("\nMean Particle Concentration/ml\n") + # Y axis label
  ggtitle("Nanosight Histogram of\nVirgin Mouse Plasma")+ #title
  labs(color="Injection")+ #Label table title
  facet_wrap( ~Injection)

nanosight_plot

## Warning: Removed 1000 rows containing missing values (geom_path).

ggsave("Nanosight_plot.png", height = 5, width = 7, units = "in", dpi = 600)

## Warning: Removed 1000 rows containing missing values (geom_path).

 test3 %>% 
  filter(!Sample_ID %in% c('1','6','15','16','19','24','29','6','28','32')) %>% 
  group_by(Day) %>% 
  ggplot(aes(x= Day, y = particle_conc, color=Day)) +
  geom_boxplot(colour="black",fill=NA) + 
  geom_point(aes(text = paste("Sample ID:", Sample_ID)),
             position='jitter',size=3)+
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nExosomes/ml\n") + # Y axis label
  ggtitle("Plasma Exosome Concentration\nThroughout Pregnancy\n")+ #title
  labs(color="Condition")+ # Label table title
  scale_x_discrete(breaks=c("1","5","10","14","17","20"),  # Change X axis label
                   labels=c("Virgin","5","10","14","17","1 Day Post")) +
  scale_color_discrete(labels=c("Virgin","5","10","14","17","1 Day Post")) # Change Legend

## Warning: Ignoring unknown aesthetics: text

 filtered_data <- test3 %>% 
  filter(!Sample_ID %in% c('1','6','15','16','19','24','29','6','28','32'))


filtered_fit <- aov(particle_conc ~ Day, data=filtered_data)
filtered_stats <- tidy(filtered_fit)
filtered_stats

filtered_HSD <- TukeyHSD(filtered_fit)
filtered_tukey <- tidy(filtered_HSD)

filtered_tukey %>% 
  filter(adj.p.value < 0.05) %>% 
  arrange(adj.p.value)

Mean placenta weight

mean_placenta %>% 
  group_by(Day) %>% 
  summarise(mean_N   = length(mean_plac),
            day_mean = mean(mean_plac),
            day_sd   = sd(mean_plac),
            day_se   = day_sd/sqrt(mean_N)) %>% 
  ggplot(aes(x = Day, y = day_mean, fill = Day))+
  geom_bar(stat="identity")+
  geom_errorbar(aes(ymin = day_mean-day_se, ymax = day_mean+day_se), width=.5, 
                size=0.8, colour="black", position=position_dodge(.9)) + #error bars
  scale_y_continuous(breaks = seq(0,125,25),
                     limits = c(0,125),
                     expand = c(0,0))+ #set bottom of graph and scale
  xlab("\nDay of Gestation\n") + # X axis label
  ylab("\nWeight (mg)\n") + # Y axis label
  ggtitle("Mouse Placental Weight\nThroughout Pregnancy\n")+ #title
  labs(fill="Gestational Day") # Label table title